Method of producing avian eggs and birds of germ-free status

ABSTRACT

The invention provides a method of rearing a bird of germ-free status. The method comprises housing a bird as a parent bird, surgically removing an egg in its shell from the parent bird prior to transfer of the egg to the cloaca in the parent bird, incubating the egg still in its shell and hatching the egg to produce a laying bird. The invention also relates to the production of avian eggs of germ-free status.

FIELD OF THE INVENTION

[0001] The present invention relates to a method of rearing a bird ofgerm-free status. It further relates to the production of avian eggs ofgerm-free status. It also relates to a method of producing various eggsand birds.

[0002] In this specification, the term “germ-free” is used very broadlyand relates to many pathogens and infections that can be carried bybirds, particularly, poultry such as chickens and turkeys which are usedwidely to produce flocks of birds for breeding to produce fertile eggsfor commercial production and to produce eggs and meat for humanconsumption. Further, such eggs and birds are used in the manufacture ofa wide range of biological substances including vaccines, fibroblastsand proteins, both for therapeutic and prophylactic use in people andanimals. Poultry, particularly hens, is used extensively for thesetasks. They are further used extensively for diagnostic tests and theproduction of transgenic eggs and birds. Many of these uses require eggsand/or the birds produced from them to be free of all contaminants suchas infections, including a variety of species of parasite, bacteria,mycoplasma, viruses, retroviruses, prions, DNA and RNA fragments.Sometimes, the viruses can be small viruses including picoma and parvoviruses. Some of the bacteria from which eggs are often contaminatedinclude Clostridia and Enterobacteria. There are many non-pathogenicorganisms that should be controlled. Similarly, many of themicro-organisms which include parasites, aerobic and anaerobic bacteria,commensal species and species associated with the gut, are undesirable.Similarly, mycoplasma, viruses including retroviruses, prions, fungi,yeast and moulds are also undesirable.

[0003] Therefore, the term “germ-free status” includes all of these andis much broader than just free of specified pathogens. For example,conventional specific pathogen free (SPF) are not specified free fromsome viruses and indeed can be contaminated with bacteria and indeed forcertain uses, these may be sufficient. The use to which the eggs and thebirds are to be put will determine the contaminants that the egg or birdmust be free of. Conventional contaminant free and some SPF eggs arederived by treating fresh naturally laid eggs with chemicals, includingdisinfectants and antibiotics, and placing them in isolators. Suchnaturally laid eggs are taken from selected parent stock birds. Whilethese methods have been relatively successful in the production of SPFeggs, they have not been truly successful in producing what aregerm-free eggs as the chemicals are not able to eliminate contaminationfrom, for example, bacteria entering the pores of the eggshellimmediately after laying and before disinfection. Contamination of eggsresults in loss of compliance with specifications and, in manyinstances, loss of commercial value and utility.

[0004] Additionally, the term “similar” is used in a broad sense andcould refer to the same species or indeed, could be not the same speciesthat had been reared together naturally for some considerable time, ormight not, for example, be the same species but might have some othersimilarity that was required. Thus, they might not be of a particularspecies but might have effectively been grown naturally over many yearsto form a flock that could be described as similar.

[0005] 1. Description of Prior Art

[0006] U.S. Pat. Specification No. 5,011,780 (Margaret Mary Perry)describes an in vitro avian embryo culture technique which, while notparticularly applicable to the present invention, describes in somedetail the embryonic development of eggs. This specification is directedto the incubation of an embryo in a closed container after the embryohas been removed from its shell. Indeed, in this specification, thecontainer used is preferably part of an egg shell which has been chosenfrom the same species as is being cultured or, in the terms of thepresent invention, from a similar hen. This invention is directedtowards the genetic engineering of poultry but also to the investigationof fundamental mechanisms of avian development. It is directed towardsproviding an alternative to surgical intervention in the laying hen.

[0007] Similarly, U.S. Pat. Specification No. 5,286,641 (Naito et al)discloses an in vitro culture method for a fertilised ovum of a hen inwhich an embryo which has just been fertilised is taken from an upperportion of the magnum of the oviduct of a hen within an hour or so afteroviposition and then subsequently cultured. However, both of thesespecifications merely disclose the artificial culturing of eggs and donot deal with the purpose of the present invention.

[0008] 2. Object of the Invention

[0009] The present invention is directed towards providing eggs ofgerm-free status to allow various diagnostic tests to be performed.Further, the invention is directed towards providing eggs and birds thatcould be used in the manufacture of a wide range of biologicalsubstances. Thus, the invention is directed towards providing both eggsand birds which are free of all contaminants such as infections,including a variety of species of parasites, bacteria, mycoplasma,viruses, retroviruses, prions, DNA and RNA fragments.

[0010] Indeed, it is an object of the present invention to produce, whatare effectively sterile eggs and hens which could be used as food, incertain specified situations for particularly delicate patients.

SUMMARY OF INVENTION

[0011] The invention provides a method of rearing a bird of germ-freestatus. The method comprises choosing a bird and housing it as a parentbird and then removing the egg from the parent bird prior to thetransfer of the egg to the cloaca in the parent bird, thus avoiding mostcontaminants to the egg that would normally occur within the parentbird. As an eggshell is porous external contamination is a majorproblem. Then, the egg is incubated in a sterile environment and hatchedto produce a laying bird. Generally speaking, these laying birds willall come from the one flock of similar birds, all reared under the sameconditions. They may, for example, be hatched naturally in a sterileenvironment. The flock may already be a flock of birds which are of aparticular contaminant free status which may have been produced, forexample, by natural selection under controlled conditions. The wholepurpose is to provide germ-free birds. Very often, therefore, samples ofthe laying birds will be removed and tested for specific contaminants toprovide a measure of the germ-free status. By an iterative process, itwill be possible to eventually produce a flock of birds which will bevirtually sterile and of a germ-free status. Thus, initially, when thelaying birds are produced, they will not form a flock of sufficientlygerm-free status and it will be necessary to continue the process untilbirds of the desired germ-free status are obtained.

[0012] In accordance with the invention, the egg is surgically removedfrom the parent bird by aseptic surgical laparotomy. Thus, the birds areeuthanased and then the surgical operation is performed.

[0013] The surgical operation, in one method according to the invention,comprises:

[0014] performing a laparotomy incision and tying off the oviduct of thebird at both ends with sutures;

[0015] transecting the oviduct distal to each suture;

[0016] removing the egg enclosed in the oviduct;

[0017] sterilising the oviduct;

[0018] removing the egg; and

[0019] sterilising the egg.

[0020] The eggs are generally removed at a time prior to and as close aspossible to the transfer time when the egg would naturally transfer tothe cloaca and thus the laying pattern of a parent bird will often berecorded over time to ensure that this is done as close to the estimatedtransfer time as possible.

[0021] Indeed, the sterility can be further improved by feeding thelaying birds, in the sterile environment, with food containing normalgutflora or sterile food. It will be appreciated that when birds arehatched which are not laying birds, they will then be retained forsubsequent fertilisation of the laying birds. In this way, the wholeflock can be sterile.

[0022] It will be possible, in the present invention, to produce simplythe eggs for subsequent use. When eggs are required of a germ-freestatus, the first thing to do is to incubate the eggs by using thedesired parent birds. Then, when the parent birds have been tested forspecified contaminants to provide a measure of the germ-free status,house that laying bird in another sterile environment and use thatlaying bird to lay eggs which will have a germ-free status.

[0023] Preferably, on laying, the outer shell of the egg is sterilised.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The invention will be more clearly understood from the followingdescription of the method according to the present invention.

[0025] Essentially, what the present invention does is to provide theuse of artificially derived eggs from parent birds in the production ofeggs and derived birds to give laying birds for the control ofmicro-organisms. The said eggs and birds are as appropriate to theirutility subsequently hatched, reared, maintained and bred, eitherconventionally, or in some form of isolator or sterile environment.

[0026] Method

[0027] Fifty adult female and five adult male chickens of known SPFstatus were maintained on selected diets and allowed to breed naturally.Timing of egg laying (oviposition) was recorded individually for eachfemale over a two-week period. The mean time of day (time, L) when anegg was laid was calculated for each female. The time of day for L-3hwas calculated and the period from L-3 to L was nominated as thederivation interval. This interval was the time in which asepticsurgical laparotomy was performed for removal of the most developed eggsin each bird.

[0028] For the procedure, birds were euthanased by cervical dislocationand shortly afterwards prepared. Birds were submerged in a disinfectantsolution for 5 minutes. Feathers were removed from the ventral thoraxand abdomen and the exposed skin sterilised using a 50% solution ofiodine in alcohol heated to 37° C. Each bird was then placed under aspecially adapted surgical isolator sterilised with a 5% solution ofperacetic acid and containing sterile instruments and a 500 ml flaskcontaining iodine in alcohol. The bird was covered with a sterile drapeand a sterile entry port of the isolator was then placed over the drape.A laparotomy incision was made and the oviduct (typically the uterus)was tied off at both sides of the egg using suture material. The oviductwas then transected distal to each of the sutures from the egg and theoviduct containing the egg was removed from the females' abdomen. Theuterus-enclosed egg was then placed in the iodine/alcohol solution forfive minutes after which the oviduct-enclosed egg was transferred via anentry port from the surgical isolator to a receiving isolator. In thereceiving isolator, the oviduct was incised, the egg removed, swabbedwith a disinfectant solution and transferred to an isolator adapted as ahatchery incubator.

[0029] Within one day of hatching, live chickens were removed from thehatchery isolator and transferred to two large-scale rearing isolatorssuitable for rearing groups of young chickens. Chickens were reared oncommercial diets sterilised by radiation. At 18 days of age, fivechickens were removed from each of the rearing isolators, euthanased andsampled for bacteriology by aerobic and anaerobic culture. Samplesincluded liver, spleen, heart blood, vagina/cloaca, caecal and smallintestinal digesta and faeces.

[0030] Results

[0031] Viable chickens were hatched successfully from the artificiallyderived eggs (hatchability>50% more often>90%). No anaerobic or aerobicbacteria were isolated from the chickens sampled.

CONCLUSION

[0032] A safe and highly effective method for artificial production ofgerm-free fertile eggs in chickens was established. Eggs were viable andproduced viable germ-free chickens which were successfully maintained inisolators.

[0033] It will be appreciated that according to the invention,essentially these are artificially or surgically derived eggs which,strictly speaking, in accordance with the present invention means thatthe egg is removed from the parent bird prior to the transfer of the eggto an area of potential contamination. Ideally, one raises the bird as aparent bird in a sterile environment, feeding the bird with sterilefood. Then, the egg is removed from the parent bird artificially priorto the transfer of the egg to an area of potential contamination in theparent bird and then the egg is incubated and hatched to produce alaying bird which is kept in this sterile environment.

[0034] Female parent birds may be either live or recently killed. Livebirds may, as consistent with ethical, legal and animal welfareconsiderations, be fully conscious, sedated or anaesthetised. Eggs andova may be either fertilised or unfertilised.

[0035] Infectious organisms that may be controlled by the inventioninclude organisms that can be pathogenic or non-pathogenic to therelevant species. These include avian species (typically chickens, fowlsand turkeys), humans and other mammals (typically dogs, cats, horses,cattle, pigs, sheep, goats, rats and mice). For the purposes of theinvention, micro-organisms include parasites, bacteria (includinganaerobic and aerobic species, commensal species and species associatedwith the gut), mycoplasma, viruses (including retroviruses), prions,fungi, yeasts, moulds and DNA and RNA fragments.

[0036] If fertile eggs are used to produce offspring or derived birds,then the eggs may be hatched, reared, maintained and bred in eitherconventional husbandry systems, germ-free systems or in isolators tocontrol the entry of micro-organisms.

[0037] According to the invention, for maximum freedom frommicro-organisms eggs should preferably be derived aseptically fromparent females (unless they are also germ-free) and the life-cycleshould be completed in isolators. The life-cycle may be completedoutside isolators when germ-free eggs and birds are produced.

[0038] According to the present invention, the aseptic derivation ofeggs and, if appropriate hatching, rearing, maintenance and breeding ofbirds may be used in combination with another method of controllingmicrobial contamination. Such methods include disinfectants,antimicrobials, antibiotics, antiviral agents, antiparasitics,immunomodulators and vaccines.

[0039] It will be appreciated that in certain circumstances, when takingselected birds as parent birds, the laying birds produced may not infact be sufficiently free of contaminants to produce laying birds of theright quality. It may then be necessary to carry out the same stepsagain using the eggs produced from such laying birds and artificiallyremoving the eggs from these laying birds to provide further layingbirds which hopefully will be germ-free.

[0040] While in the above, the description has related entirely topoultry and specifically hens, it will be appreciated that the presentinvention may be carried out on other birds.

[0041] In the specification the terms “comprise, comprises, comprisedand comprising” or any variation thereof and the terms “include,includes, included and including” or any variation thereof areconsidered to be totally interchangeable and they should all be affordedthe widest possible interpretation and vice versa.

[0042] The invention is not limited to the embodiment and methodsdescribed above, but may be varied within the scope of the claims.

1-16. (canceled)
 17. A method of providing an egg of a specifiedgerm-free status in a sterile environment comprising the steps of:— (a)providing a bird of germ-free status comprising the steps of: housing abird as a parent bird; removing an egg in its shell from the parent birdprior to transfer of the egg to the cloaca in the parent bird; andincubating the egg in its shell and hatching the egg to produce a layingbird; (b) housing the laying bird; (c) using the laying bird to lay theegg; and (d) removing the egg to another sterile environment.
 18. Themethod according to claim 17, in which the egg is, on laying,immediately removed and the shell of the egg is sterilized.
 19. An eggproduced in accordance with the method of claim
 17. 20. A bird hatchedfrom an egg as claimed in claim
 17. 21. A bird hatched in accordancewith the method of claim
 17. 22. A bird hatched from an egg laid by alaying bird reared in accordance with the method of claim
 17. 23-35.(canceled)
 36. A method of providing an egg of a germ-free status in asterile environment comprising the steps of:— (a) providing a bird ofgerm-free status comprising the steps of: obtaining a flock of similarbirds all reared under the same conditions; housing one of the birds asa parent bird; removing an egg from the parent bird prior to transfer ofthe egg to the cloaca in the parent bird; and incubating the egg in itsshell and hatching the egg to produce a laying bird; (b) housing thelaying bird; (c) using the laying bird to lay the egg; and (d) removingthe egg to another sterile environment.
 37. The method according toclaim 36, in which the egg is, on laying, immediately removed and theshell of the egg is sterilized.
 38. An egg produced in accordance withthe method of claim
 36. 39. A bird hatched from an egg as claimed inclaim
 36. 40. A bird hatched in accordance with the method of claim 36.41. A bird hatched from an egg laid by a laying bird reared inaccordance with the method of claim
 36. 42-46. (canceled)
 47. A methodof providing an egg of a germ-free status in a sterile environmentcomprising the steps of:— (a) providing a bird of germ-free statuscomprising the steps of: obtaining a flock of similar birds all rearedunder the same conditions; housing one of the birds as a parent bird;performing a laparotomy incision and tying off the oviduct of the birdat both ends with sutures prior to transfer of the egg to the cloaca inthe parent bird; transecting the oviduct distal to each suture; removingthe egg in its shell enclosed in the oviduct; sterilizing the oviduct;removing the egg; sterilizing the exterior of the egg shell; andincubating the egg and hatching the egg to produce a laying bird; (b)housing the laying bird; (c) using the laying bird to lay the egg; and(d) removing the egg to another sterile environment.
 48. The methodaccording to claim 47, in which the egg is, on laying, immediatelyremoved and the shell of the egg is sterilized.
 49. An egg produced inaccordance with the method of claim
 48. 50. A bird hatched from an eggas claimed in claim
 48. 51. A method of providing an egg of a germ-freestatus comprising the steps of, in a sterile environment:— housing abird as a parent bird; removing an egg from the parent bird prior totransfer of the egg to the cloaca in the parent bird; incubating the eggand hatching the egg to produce a laying bird; testing the laying birdfor specific contaminants to provide a measure of the germ-free status;housing the laying bird in another sterile environment; using the layingbird to lay the egg; and removing the egg to another sterileenvironment.
 52. The method according to claim 51, in which the bird ischosen from a flock of similar birds, all reared under the sameconditions and the laying bird produced is used to form a flock of birdsof germ-free status.
 53. The method according to claim 51, in whichlaying bird forms part of a flock and after the laying birds arehatched, a sample of the laying birds is removed and tested for specificcontaminants to provide a measure of the germ-free status of the flock.54. The method according to claim 51, in which the laying bird formspart of a flock and after the laying birds are hatched, a sample of thelaying birds is removed and tested for specific contaminants to providea measure of the germ-free status of the flock and in which when thegerm-free status is not achieved in the laying bird, the laying bird isused as a parent bird in the method.
 55. The method according to claim51, in which the egg is surgically removed from the parent bird andcomprises the steps of:— performing a laparotomy incision and tying offthe oviduct of the bird at both ends with sutures; transecting theoviduct distal to each suture; removing the egg enclosed in the oviduct;sterilizing the oviduct; removing the egg; and sterilizing the egg. 56.The method according to claim 51, in which the egg is, on laying,immediately removed and the shell of the egg is sterilized.
 57. A birdhatched from an egg as claimed in claim
 51. 58. A method of providing anegg of a specified germ-free status comprising the steps of: (a)providing a bird of germ-free status comprising the steps of: housing abird as a parent bird; in a sterile environment removing an egg in itsshell from the parent bird prior to transfer of the egg to the cloaca inthe parent bird; and in a sterile environment incubating the egg in itsshell and hatching the egg to produce a laying bird; (b) housing thelaying bird in a sterile environment; and (c) using the laying bird tolay the egg in a sterile environment.
 59. The method according to claim58, in which the egg is, on laying, immediately removed and the shell ofthe egg is sterilized.
 60. An egg produced in accordance with the methodof claim
 58. 61. A bird hatched from an egg as claimed in claim
 58. 62.A bird hatched in accordance with the method of claim
 58. 63. A birdhatched from an egg laid by a laying bird reared in accordance with themethod of claim
 58. 64. A method of providing an egg of a germ-freestatus comprising the steps of: (a) providing a bird of germ-free statuscomprising obtaining a flock of similar birds all reared under the sameconditions; housing one of the birds as a parent bird; and in a sterileenvironment removing an egg from the parent bird prior to transfer ofthe egg to the cloaca in the parent bird; in a sterile environmentincubating the egg in its shell and hatching the egg to produce a layingbird; (b) housing the laying bird in a sterile environment; and (c)using the laying bird to lay the egg in a sterile environment.
 65. Themethod according to claim 64, in which the egg is, on laying,immediately removed and the shell of the egg is sterilized.
 66. An eggproduced in accordance with the method of claim
 64. 67. A bird hatchedfrom an egg as claimed in claim
 64. 68. A bird hatched in accordancewith the method of claim
 64. 69. A bird hatched from an egg laid by alaying bird reared in accordance with the method of claim
 64. 70. Amethod of providing an egg of a germ-free status comprising the stepsof: (a) providing a bird of germ-free status comprising the steps of:obtaining a flock of similar birds all reared under the same conditions;housing one of the birds as a parent bird; in a sterile environmentperforming a laparotomy incision and tying off the oviduct of the birdat both ends with sutures prior to transfer of the egg to the cloaca inthe parent bird; transecting the oviduct distal to each suture in asterile environment; removing the egg in its shell enclosed in theoviduct in a sterile environment; sterilizing the oviduct; removing theegg in a sterile environment; and sterilizing the exterior of the eggshell; (b) incubating the egg and hatching the egg to produce a layingbird in a sterile environment; (c) housing the laying bird in a sterileenvironment; and (d) using the laying bird to lay the egg in a sterileenvironment.
 71. The method according to claim 70, in which the egg is,on laying, immediately removed and the shell of the egg is sterilized.72. An egg produced in accordance with the method of claim
 71. 73. Abird hatched from an egg as claimed in claim
 71. 74. A method ofproviding an egg of a germ-free status comprising the steps of: housinga bird as a parent bird; in a sterile environment removing an egg fromthe parent bird prior to transfer of the egg to the cloaca in the parentbird; incubating the egg and hatching the egg to produce a laying birdin a sterile environment; testing the laying bird for specificcontaminants to provide a measure of the germ-free status; housing thelaying bird in a sterile environment; using the laying bird to lay theegg in a sterile environment; and removing the egg to another sterileenvironment.
 75. The method according to claim 74, in which the bird ischosen from a flock of similar birds, all reared under the sameconditions and the laying bird produced is used to form a flock of birdsof germ-free status.
 76. The method according to claim 74, in whichlaying bird forms part of a flock and after the laying birds arehatched, a sample of the laying birds is removed and tested for specificcontaminants to provide a measure of the germ-free status of the flock.77. The method according to claim 74, in which the laying bird formspart of a flock and after the laying birds are hatched, a sample of thelaying birds is removed and tested for specific contaminants to providea measure of the germ-free status of the flock and in which when thegerm-free status is not achieved in the laying bird, the laying bird isused as a parent bird in the method.
 78. The method according to claim74, in which the egg is surgically removed from the parent bird andcomprises the steps of:— performing a laparotomy incision and tying offthe oviduct of the bird at both ends with sutures; transecting theoviduct distal to each suture; removing the egg enclosed in the oviduct;sterilizing the oviduct; removing the egg; and sterilizing the egg. 79.The method according to claim 74, in which the egg is, on laying,immediately removed and the shell of the egg is sterilized.
 80. A birdhatched from an egg as claimed in claim 74.